Genes for resistance to and biodegradation of organomercurials (Omr) including the antiseptic merthiolate, the disinfectant phenylmercuric acetate, and the neurotoxic fungicide methyl mercury, are often found on bacterial plasmids along with resistance to inorganic compounds such as HgCl2. Though inorganic mercury resistance (Hgr) has been studied extensively, much less is known about organomercury resistance. Two years ago we began to study the organomercury resistance determined by the conjugative IncM plasmid, R831b. Though 90 kb in size, R831b is the smallest naturally occurring plasmid which carries the Omr locus. Using transposon mutagenesis and restriction enzyme analysis we have mapped the only two selectable markers carried by R831b, Hgr and Omr. We found that these loci are separated by 13.5 kb despite the fact that the enzymes they encode are regulated co-ordinately. We have sub-cloned Omr into pBR322 which is compatible with the Hgr mini-IncM replicon we had previously cloned and have begun to construct fine structure restriction and deletion maps of both loci. During the last year of this present grant we will complete the restriction enzyme mapping of these loci and generate both lac and galK fusions in the parental plasmid and in sub-cloned derivatives. We propose for the long term: (1) to use minicell polypeptide analysis to characterize the gene products of the Omr and Hgr loci of R831b; (2) to examine regulation of Omr/Hgr expression using insertion and deletion mutants and lac and galK fusions of both the homologous system (i.e. the R831b loci) as well as of a heterologous system (i.e. including both R831b and the correlate loci of the mer operon of NR1) and to isolate the cis- and trans-acting regulatory elements of these loci in R831b and compare them with those of the NR1 mer operon; (3) to employ the Omr and Hgr DNA as probes to explore the occurrence of such loci in our large collection of multiply metal resistant bacteria; and (4) to sequence the Omr and Hgr loci of R831b. No Omr locus has yet been sequenced and though Hgr loci from plasmid NR1 and the related transposon Tn501 have been sequenced, our polypeptide data, DNA hybridization data (ours unpublished and T. Barkay, personal communication) and immunodiffusion data (S. Silver, personal communication) indicate that the Hgr locus of R831b is not closely related to that of NR1/Tn501.